1. Field of the Invention
The present invention is concerned with improved colorimetric or fluorometric systems useful for developing characteristic colors or fluorescence in the presence of a peroxidase enzyme, or a peroxidase enzyme-linked moiety. More particularly, it is concerned with such systems and methods of use thereof, which utilize in the substrate an accelerator for enhancing the colorimetric or fluorometric reaction(s). Broadly speaking, the accelerator should be selected from the group consisting of substituted phenol compounds, and should provide significant rate enhancement when compared to identical, accelerator-free substrates.
2. Description of the Prior Art
The peroxidase enzymes, and particularly horseradish peroxidase, have become the enzymes of choice in many enzyme-linked immunoassay systems. Horseradish peroxidase is extremely stable, has high substrate turnover rate, and is able to yield both chromogenic and fluorogenic products from a variety of different substrates. The chromogenic substrates have proven to be ideal for visual qualitative determinations, while both types of substrates have found diverse applications in instrument monitored quantitative determinations. See Worthington Enzymes, Worthington Biochemical Corp., Freehold, N. J.; K. G. Paul (1963), The Enzymes, Vol. 9, Part B, Chapter 7, Academic Press, New York; and H. S. Mason, Advances In Enzymol, (1957) 19, 79.
Although perhaps the most widespread use of horseradish peroxidase is in conjunction with ELISA (enzyme linked immunoassay) determinations, its use has not been so limited. In fact, horseradish peroxidase can also be used in coupled assays for the detection and determination of glucose, galactose and certain amino acids in conjunction with their respective oxidases.
In those systems where a peroxidase enzyme is employed as a tag or label, the final determination, be it either qualitative or quantitative, is made either colorimetrically or fluorometrically. Typically, this involves reacting the enzyme with a colorimetric or fluorometric substrate which would normally include a peroxide type oxidizing agent, a compound capable of reacting and giving off color or fluorescence in the presence of the enzyme and the oxidizing agent, and a buffering system.
The net reaction of horseradish peroxidase (HRPO) in the presence of a normal substrate including a chromogenic or fluorometric compound (AH.sub.2) may be represented schematically by: ##STR1## The primary products are radicals which react in solution and may form chromogenic or fluorogenic final products. Normally .sup.k 7 is much greater than .sup.k 4 and therefore the second electron abstraction is rate limiting. (B. Chance, Arch. Biochem. Biophy (1952), 41, 404, ibid. p. 416). Both reaction 2 and 3 above involve the transfer of a single electron from the substrate to the enzyme. (P. George, Nature (1952), 169, 612; B. Chance, Arch. Biochem. Biophy (1952), 41, 404, ibid. p. 416). .sup.k 1 is rate limiting only when hydrogen peroxide is present in limiting amount, and therefore any compound affecting the apparent rate probably affects .sup.k 4, i.e., the second electron abstraction. In any system employing a substrate of the type described above, any factor or catalyst that would accelerate the rate of product formation would effectively permit shorter assay times and increased sensitivity. That is to say, in an enzyme-linked immunoassay, the doubling of the rate would allow for interpretation of results in half the previous incubation time. A logical extension of this argument is that if time is held constant, then the test system should be able to detect half the amount of analyte previously detectable. Similar considerations apply to the coupled reactions utilizing unlinked horseradish peroxidase.
A number of assay conditions have been previously described which can cause such a desirable increase in enzyme activity. Compounds known to increase horseradish peroxidase activity include nitrogenous ligands (I. Fridovich, J. Biol. Chem. (1963), 238, 3921), palmitic acid (A. K. Mattoo and V. V. Modi (1975) Biochemica Biophys Acta 397, 381), and non-ionic detergents (B. Porstmann, et al. (1981) Clinica Chem Acta, 109, 175). In 1963, Fridovich demonstrated that the nitrogenous ligands ammonia, pyridine and imidazole increase the rate of peroxidation of dianisidine by horseradish peroxidase. In subsequent studies, Claiborne and Fridovich have suggested that the mechanism for this acceleration involves the nucleophilic base facilitating the abstraction of a second electron from the substrate radical intermediate. (Biochem (1979), 18, 2329). These authors also suggested that the 2 electron abstractions, as 2 distinct steps, is the true mechanism that occurs in the peroxidase reaction. If this proposed mechanism is correct the free-radical is bound to the enzyme and only released after a second electron is abstracted with the intermediate rearranging to form the product. However, this proposed divalent abstraction is at variance with other univalent and simple divalent proposals (B. Chance 1952, P. George, 1952, R. Roman and H. B. Dunford, Biochem (1972) 11, 2076, R. Roman and H. B. Dunford, Can. J. Chem. (1973) 51, 588). But the mechanism proposed by Claiborne & Fridovich, see the single electron abstraction described above (P. George, 1952, B. Chance, 1952), should be evaluated with caution for it is based on data collected with horseradish peroxidase catalyzed peroxidation of o-dianisidine and p-phenylenediamine two compounds known to participate in reversible two-electron oxidations (Piette et al., Ana. Chem. (1962), 34, 916). Fridovich (1963) also reported that peroxidation kinetics observed with o-dianisidine and p-phenylenediamine were not found with other horseradish peroxidase substrates. This may suggest that double electron abstraction occurs only where the substrate can easily undergo a double oxidation.
In addition to nitrogenous ligands, palmitic acid has been demonstrated to increase the rate of horseradish peroxidase peroxidation of o-dianisidine (Mattoo and Modi, 1975). But activation by palmitic acid occurs only at low substrate concentrations and may have no significant effect in analytical systems, such as enzyme-linked immunoassays, where substrate is present in great excess. However, the palmitic acid dependent activation may be of utility in oxidase coupled reactions where substrate depletion does occur. By comparison, while, palmitic acid activation of horseradish peroxidase appears to be of limited usefulness, the activation of horseradish peroxidase by non-ionic detergents has greater applicability.
The commercially available non-ionic detergents Tween 20 and Triton X-100 were demonstrated to increase the peroxidation of a number of different substrates by Porstmann, et al. (1981). In this system the analytical sensitivity in an enzyme-immunoassay was approximately doubled by the addition of non-ionic detergent. The non-ionic detergent dependent increase in activity is the result of decreased inactivation of horseradish peroxidase. The time and temperature dependent inactivation is possibly the result of formation of a terminal complex between hydrogen peroxide and enzyme, (H. Gallati, J. Clin. Chem. Clin. Biochem. (1977), 15, 699). This clearly illustrates a point noted above, i.e., by increasing the rate, Fridovich (1963), with ammonium ligands or maintenance of a rate, Porstmann, et al. (1981) with Tween 20, an increased analytical sensitivity is possible.